1. Field of the Invention
The present invention relates to specific inhibitors of heparanase catalytic activity, directed to an amino acid sequence derived from the N-terminus sequence of the 50 Kd cleavage product of heparanase precursor. More particularly, the invention relates to an amino acid sequence derived from amino acid residues Lys158-Asp171 of human heparanase and any functional fragments thereof, and the use of said sequence in a screening method for specific heparanase inhibitors. The invention further provides compositions and methods comprising said heparanase inhibitors for the treatment of heparanase related disorders.
2. Prior Art
Throughout this application various publications are referenced to. It should be appreciated that the disclosure of these publications in their entireties are hereby incorporated into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
Heparanase is an endo-β-D-glucuronidase involved in cleavage of heparan sulfate (HS) chains, and hence participates in extracellular matrix (ECM) degradation and remodeling. Heparanase activity has been traditionally correlated with the metastatic potential of tumor-derived cell types [Nakajima, M. et al., J. Cell. Biochem. 36, 157-167 (1998); Vlodavsky, I. et al., Nat. Med. 5, 793-802 (1999); Parish, C. R. et al., Biochem. Biophys. Acta 1471, M99-M108 (2001); Vlodavsky, I. and Friedmann Y., J. Clin. Invest. 108, 341-347 (2001)]. Similarly, heparanase has been shown to facilitate cell invasion associated with autoimmunity, inflammation and angiogenesis [Vlodavsky, I. et al., Invasion & Metastasis 12, 112-127 (1992); Dempsey, L. et al., Trends Biol. Sci. 25, 349-351 (2000a); Parish (2001) ibid.]. More recently, heparanase upregulation was detected in a variety of human primary tumors correlating, in some cases, with increased tumor vascularity and poor postoperative survival [El-Assal, O. N. et al., Clin. Cancer Res. 7, 1299-1305 (2001); Gohji, K. et al., Int. J. Cancer 95, 295-301 (2001); Koliopanos, A. et al., Cancer Res. 61, 4655-4659 (2001); Rohloff, J. et al., J. Cancer 86, 1270-1275 (2002)]. In addition, increased heparanase expression has been noted in kidney [Levidiotis, V. et al., Kidney Int. 60, 1287-1296 (2001)], liver [Xiao, Y. et al., Hepatology Res. 26, 192-198 (2003)] and diabetic [Katz, A. et al., Isr. Med. Assoc. 4, 996-1002 (2002)] disorders. In the latter case, increased heparanase activity in patient's urine has been detected, suggesting that heparanase may serve as an early marker in diabetes and potentially other pathologies such as tumor metastasis [Goldshmidt, O. et al., Proc. Natl. Acad. Sci. USA 99, 10031-10036 (2002)]. Increased heparanase activity in urine and possibly other body fluids strongly implies that heparanase is a secreted enzyme. In addition, exogenously added and endogenous heparanase were localized to endosomes and lysosomes [Nadav, L. et al., J. Cell Sci. 115, 2179-2187 (2001); Goldshmidt (2002) ibid.].
The heparanase cDNA encodes for a polypeptide of 543 amino acids that appears as a ˜65 kDa protein in SDS-PAGE analysis. The protein undergoes proteolytic processing which is likely to occur at two potential cleavage sites, Glu109-Ser110 and Gln157-Lys158, yielding an 8 kDa polypeptide at the N-terminus, a 50 kDa polypeptide at the C-terminus and a 6 kDa linker polypeptide that resides in-between [Fairbanks, M. B. et al., J. Biol. Chem. 274, 29587-29590 (1999); Parish (2001) ibid.]. Recently published observations clearly demonstrated that the active heparanase enzyme exists as a heterodimer composed of the 8 kDa polypeptide non-covalently associated with the 50 kDa heparanase subunit, and that heterodimer formation is necessary and sufficient for heparanase enzymatic activity [Levy-Adam, F. et al., Biochem. Biophy. Res. Comm. 308, 885-891 (2003); McKenzie, E. et al., Biochemical J. 373, 423-435 (2003)]. Nevertheless, currently available anti-heparanase antibodies do not distinguish between the latent 65 kDa heparanase precursor and the 50 kDa active enzyme. Thus, specific localization of the latent and active heparanase forms within the cell could not be determined.